Dengue virus, a flavivirus, causes human disease throughout subtropical and tropical regions of the world. There are four serotypes; infection with one produces durable immunity only to that type. Serotype is specified by the viral structural proteins. The dengue viral genome, an 11 kilobase strand of positive-sense RNA, contains of three structural protein genes followed by a series of 7 non-structural protein genes. Earlier, we constructed a full-length cDNA copy of the entire dengue 4 (D4) genome, RNA transcripts from which were infectious when transfected into mammalian cells. We subsequently replaced the structural protein genes of the full- length clone with the structural protein genes of D1 or D2 virus, and used these templates to create chimeric D1/4 and D2/4 viruses. The D4/D1 chimeric virus have been made, one using a D2 virus isolated from a human patient, the other a mutant of that virus selected for mouse neurovirulence through serial mouse brain passage. The chimeric D4/neurovirulent D2 virus retained the neurovirulence property of its parent, but the level of neurovirulence was less than that of the parental type 2 virus. This indicated that at least some of the genetic loci responsible for neurovirulence are located in the structural protein genes. Sequencing of these genes in the neurovirulent D2 mutant and in its non-neurovirulent parent revealed 7 mutations resulting in amino acid changes. In order to determine which mutation(s) cause neurovirulence, I plan to make a series of chimeric viruses in which these mutations are isolated against a background of the prototype sequence. Elimination of the neurovirulence property may result in a chimeric D4/D2 virus safe for humans. Since the D4/D2 chimeric viruses appear to replicate more slowly than the parental viruses, they may also have properties of attenuation desirable in a vaccine. A set of suitably engineered chimeric viruses might serve together as a safe and effective tetravalent live dengue virus vaccine.